Embryonic stem cells (ES cells) were first derived in 1981 from the inner cell mass (ICM) of murine pre-implantation blastocyst embryos (Evans and Kaufman, 1981; Martin, 1981). ES cells are pluripotent, meaning they demonstrate the ability to expand indefinitely in vitro while retaining the capacity to generate ectoderm-, endoderm-, and mesoderm derivatives both in vitro and in vivo. The discovery of murine ES cells was a major breakthrough in developmental biology, since it allowed the study of mammalian gene function in vivo, through the application of transgenic and knockout technologies. The subsequent derivation of human ES cells raised the expectation that these cells would similarly revolutionize our insights into human development and disease. Unfortunately, human pluripotent stem cells have proven to be remarkably resilient to non-viral genetic manipulation and to date only a handful of human transgenic cell lines exist.
Homologous recombination in human cells, including human ES cells is remarkably inefficient. WO2009/152529 describes preliminary data regarding the targeting of two endogenous loci, OCT4 and COLIA1 by introducing sequences encoding GFP and puromycin resistance. The authors note that so far only genes that are expressed in ES cells (OCT4, HPRT, ROSA26) or that are poised to be expressed, such as MOXL1, have been targeted in human ES cells. The authors also note that the COL1A1 locus is highly recombinogenic in mouse cells, and that targeting of that locus may not be representative of other non-expressed genes. The authors state that the targeting of non-expressed genes by homologous recombination poses a challenge.